ExoU is an acutely cytotoxic protein that is secreted by Pseudomonas aeruginosa via a type III secretion system directly into mammalian cells. P. aeruginosa is an opportunistic pathogen that infects immunocompromised individuals and patients with cystic fibrosis. In cystic fibrosis patients the expression of ExoU has been correlated with damage to the lungs and ability of the bacteria to disseminate to the blood stream. The mechanism of cytotoxicity of ExoU is unknown. Previous studies have shown that secretion of ExoU requires a specific chaperone, SpcU. Determination of the crystal structure of ExoU, alone and bound to it's chaperone, as described in this proposal, will help to understand the function of ExoU and the important role of chaperones in type III secretion. The structural studies combined with the proposed studies of the enzymatic activity of ExoU will help to determine the mechanism of cytotoxicity of this protein.